Preventives or remedies for arthritis

ABSTRACT

The present invention relates to pharmaceuticals, foods and drinks, food additives, animal feeds and feed additives comprising an N-acylated hydroxyproline derivative or a salt thereof, and an amino sugar or a salt thereof and/or a glycosaminoglycan or a salt thereof as active ingredients, use of an Nacylated hydroxyproline derivative or a salt thereof for the production of an arthritis preventing or treating agent, and a method for preventing or treating arthritis which comprises administering an N-acylated hydroxyproline derivative or a salt thereof, and an amino sugar or a salt thereof and/or a glycosaminoglycan or a salt thereof.

TECHNICAL FIELD

The present invention relates to pharmaceuticals, foods and drinks, foodadditives, animal feeds and feed additives having an effect on theprevention or the treatment of arthritis.

BACKGROUND ART

With changes in our lifestyle and aging of the population, arthritis isexpected to increase in the future.

N-Acetylhydroxyproline is known as a chemical substance that showsanti-inflammatory activity (U.S. Pat. Nos. 3,891,765 and 3,932,638;Japanese Published Examined Patent Application No. 43947/72).

It is known that administration of N-acetylhydroxyproline to an animalmodel for arthritis after induction of arthritis can prevent aggravationof arthritis [J. Drug. Dev., 3, 135–142 (1990)]. It is also known thatalthough N-acetylhydroxyproline shows a therapeutic effect on systemicinjury of ear and tail, it shows no effect on chondral injury at joints[Pharmacological Research, 33, 367–373 (1996)].

However, it has not been known so far that N-acylated hydroxyprolinederivatives such as N-acetylhydroxyproline show a preventive effect onarthritis.

Furthermore, although chondroitin, glucosamine, etc. that are thecomponents of cartilages are known to have a therapeutic effect onarthritis (WO94/22453), there has not been known a composition obtainedby the addition of an N-acylated hydroxyproline derivative thereto.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a pharmaceutical, afood and drink, a food additive, an animal feed and a feed additivehaving an effect on the prevention or the treatment of arthritis and amethod for preventing or treating arthritis in humans or non-humananimals using them.

The present invention relates to the following (1)–(22).

(1) A pharmaceutical which comprises an N-acylated hydroxyprolinederivative or a salt thereof, and an amino sugar or a salt thereofand/or a glycosaminoglycan or a salt thereof.

(2) The pharmaceutical according to the above (1), wherein saidhydroxyproline is selected from the group consisting ofcis-4-hydroxy-L-proline, cis-4-hydroxy-D-proline,cis-3-hydroxy-L-proline, cis-3-hydroxy-D-proline,trans-4-hydroxy-L-proline, trans-4-hydroxy-D-proline,trans-3-hydroxy-L-proline and trans-3-hydroxy-D-proline.

(3) The pharmaceutical according to the above (1) or (2), wherein theacyl moiety of said N-acylated hydroxyproline derivative is an acylgroup having 2–23 carbon atoms.

(4) The pharmaceutical according to any one of the above (1) to (3),wherein said N-acylated hydroxyproline derivative isN-acetylhydroxyproline.

(5) The pharmaceutical according to any one of the above (1) to (4),wherein said amino sugar is glucosamine or a salt thereof.

(6) The pharmaceutical according to any one of the above (1) to (5),wherein said glycosaminoglycan is chondroitin sulfate or a salt thereof.

(7) The pharmaceutical according to any one of the above (1) to (6),wherein said pharmaceutical is a pharmaceutical for preventing ortreating arthritis.

(8) The pharmaceutical according to the above (7), wherein saidarthritis is rheumatoid arthritis.

(9) A food and drink or an animal feed which comprises an N-acylatedhydroxyproline derivative or a salt thereof, and an amino sugar or asalt thereof and/or a glycosaminoglycan or a salt thereof.

(10) The food and drink or the animal feed according to the above (9),wherein said hydroxyproline is selected from the group consisting ofcis-4-hydroxy-L-proline, cis-4-hydroxy-D-proline,cis-3-hydroxy-L-proline, cis-3-hydroxy-D-proline,trans-4-hydroxy-L-proline, trans-4-hydroxy-D-proline,trans-3-hydroxy-L-proline and trans-3-hydroxy-D-proline.

(11) The food and drink or the animal feed according to the above (9) or(10), wherein the acyl moiety of said N-acylated hydroxyprolinederivative is an acyl group having 2–23 carbon atoms.

(12) The food and drink or the animal feed according to any one of theabove (9) to (11), wherein said N-acylated hydroxyproline derivative isN-acetylhydroxyproline.

(13) The food and drink or the animal feed according to any one of theabove (9) to (12), wherein said amino sugar is glucosamine or a saltthereof.

(14) The food and drink or the animal feed according to any one of theabove (9) to (13), wherein said glycosaminoglycan is chondroitin sulfateor a salt thereof.

(15) A food additive or a feed additive which comprises an N-acylatedhydroxyproline derivative or a salt thereof, and an amino sugar or asalt thereof and/or a glycosaminoglycan or a salt thereof.

(16) The food additive or the feed additive according to the above (15),wherein said hydroxyproline is selected from the group consisting ofcis-4-hydroxy-L-proline, cis-4-hydroxy-D-proline,cis-3-hydroxy-L-proline, cis-3-hydroxy-D-proline,trans-4-hydroxy-L-proline, trans-4-hydroxy-D-proline,trans-3-hydroxy-L-proline and trans-3-hydroxy-D-proline.

(17) The food additive or the feed additive according to the above (15)or (16), wherein the acyl moiety of said N-acylated hydroxyprolinederivative is an acyl group having 2–23 carbon atoms.

(18) The food additive or the feed additive according to any one of theabove (15) to (17), wherein said N-acylated hydroxyproline derivative isN-acetylhydroxyproline.

(19) The food additive or the feed additive according to any one of theabove (15) to (18), wherein said amino sugar is glucosamine or a saltthereof.

(20) The food additive or the feed additive according to any one of theabove (15) to (19), wherein said glycosaminoglycan is chondroitinsulfate or a salt thereof.

(21) Use of an N-acylated hydroxyproline derivative or a salt thereof,and an amino sugar or a salt thereof and/or a glycosaminoglycan or asalt thereof for the production of an arthritis preventing or treatingagent.

(22) A method for preventing or treating arthritis in humans ornon-human animals, which comprises administering an N-acylatedhydroxyproline derivative or a salt thereof, and an amino sugar or asalt thereof and/or a glycosaminoglycan or a salt thereof.

Hydroxyproline widely occurs in nature as a major amino acid componentof collagen and as an amino acid component of elastin. It is known thatthere exist eight kinds of stereoisomers of natural hydroxyproline,which are distinct in the following points: proline is the D-form or theL-form, the hydroxyl group is at the 3-position or the 4-position, andthe stereoisomer is the cis-form or the trans-form, examples thereofbeing cis-4-hydroxy-L-proline, cis-4-hydroxy-D-proline,cis-3-hydroxy-L-proline, cis-3-hydroxy-D-proline,trans-4-hydroxy-L-proline, trans-4-hydroxy-D-proline,trans-3-hydroxy-L-proline and trans-3-hydroxy-D-proline.

Although hydroxyproline of any such structure can be used in the presentinvention, trans-4-hydroxy-L-proline is preferably used.

Hydroxyproline can be obtained by subjecting collagen derived fromanimals such as pig and cow to acid hydrolysis and purifying thehydrolysate according to a conventional method. However, hydroxyprolineproduced using microorganisms is preferably used.

Useful microorganisms include those belonging to the genus selected fromAmycolatopsis, Dactylosporangium and Streptomyces or those into which aproline 3-hydroxylase gene or a proline 4-hydroxylase gene derived fromthese microorganisms has been introduced.

Introduction of a proline 3-hydroxylase gene or a proline 4-hydroxylasegene derived from a microorganism belonging to the genus selected fromAmycolatopsis, Dactylosporangium and Streptomyces into a microorganismcan be carried out according to the methods described in MolecularCloning, A Laboratory Manual, Second Edition, Cold Spring HarborLaboratory Press (1989), Current Protocols in Molecular Biology, JohnWiley & Sons (1987–1997), etc.

Furthermore, trans-4-hydroxy-L-proline can be produced using proline4-hydroxylase isolated from a microorganism belonging to the genusAmycolatopsis or Dactylosporangium (Japanese Published Unexamined PatentApplication No. 313179/95), and cis-3-hydroxy-L-proline can be producedusing proline 3-hydroxylase isolated from a microorganism belonging tothe genus Streptomyces (Japanese Published Unexamined Patent ApplicationNo. 322885/95) [Bioindustry, 14, 31 (1997)].

The acyl moiety of the N-acylated hydroxyproline derivatives used in thepresent invention includes straight-chain or branched acyl groups having2–23 carbon atoms, for example, acetyl, propionyl, butyryl, isobutyryl,valeryl, hexanoyl, heptanoyl, octanoyl, decanoyl and eicosanoyl, amongwhich acetyl and propionyl are preferred.

The N-acylated hydroxyproline derivatives can be produced according to aknown method.

That is, the N-acylated hydroxyproline derivatives can be prepared byN-acylating hydroxyproline in an aqueous medium or an organic solventusing an active derivative (acid anhydride, acid chloride, etc.) of afatty acid having an alkyl group having preferably 1–22 carbon atoms.

The N-acylated hydroxyproline derivatives thus obtained can be purifiedby conventional purification methods such as crystallization andchromatography.

Examples of the salts of N-acylated hydroxyproline derivatives includealkali metal salts such as sodium salt and potassium salt, alkalineearth metal salts such as magnesium salt and calcium salt, ammoniumsalts such as ammonium salt and tetramethylammonium salt, and organicamine addition salts such as salts with morpholine and piperidine.

In the present invention, examples of the amino sugars or salts thereofare glucosamine, galactosamine, neuraminic acid, N-acetylglucosamine,N-acetylgalactosamine, N-acetylneuraminic acid and N-glycolyl-neuraminicacid or salts thereof. Glucosamine or salts thereof are preferably used.Examples of the salts of amino sugars are hydrochloride, sulfate andphosphate.

Glucosamine obtained, for example, by hydrolyzing, with concentratedhydrochloric acid, chitin obtained by deproteinizing and decalcifyingshells of crustacean and then deacetylating, bleaching, filtering,concentrating, separating, washing and drying the hydrolysate may beused. Otherwise, commercially available products (for example,Glucosamine KHF: KYOWA HI FOODS CO., LTD.) may also be used.

Examples of the salts of glucosamine are hydrochloride, sulfate (forexample, glucosamine hexasulfate) and phosphate (for example,glucosamine hexaphosphate).

Galactosamine obtained, for example, by subjecting chondroitin sulfateprepared from cartilages of bronchus, nose, etc. of large animals andcartilages of selachian to acid hydrolysis and separating and purifyingthe resulting hydrolysate according to methods such as ion exchangechromatography may be used. Otherwise, commercially available productsmay also be used.

Examples of the salts of galactosamine are hydrochloride, sulfate (forexample, galactosamine hexasulfate) and phosphate (for example,galactosamine hexaphosphate).

Neuraminic acid commercially available as N-acetylneuraminic acid whichis an N-acylated derivative thereof and N-glycolyl-neuraminic acid whichis an N-glycolyl derivative thereof may be used.

In the present invention, the glycosaminoglycans include hyaluronicacid, chondroitin, chondroitin sulfate, keratan sulfate, heparin,heparan sulfate and dermatan sulfate and salts thereof, and chondroitinsulfate or salts thereof are preferably used.

Examples of the salts of glycosaminoglycans are sodium salt, potassiumsalt and calcium salt.

Examples of the salts of chondroitin sulfate are sodium salt, potassiumsalt and calcium salt, and sodium salt is generally used.

Chondroitin sulfate is a kind of mucopolysaccharides that are generallydistributed in connective tissues of animals, mainly in cartilages. Inthe tissues, this substance connects with protein to occur asproteoglycan.

Chondroitin sulfate to be used may be either in the form of a purifiedproduct or in the form of proteoglycan, or an extract or a dry powder ofcartilages.

Chondroitin sulfate can be obtained in the form of proteoglycan by, forexample, subjecting cartilages of aquatic animals such as shark andwhale, mammals such as cow and pig or birds as a raw material toextraction according to known methods such as neutral salt method,alkaline method, enzymatic method and autoclave method, and drying theextract after removing fat and solid content therefrom. Furthermore,after removal of fat and solid content, chondroitin sulfate or a saltthereof can be obtained in a purified form by deproteonizing theresulting extract using a protease and purifying the protein-freeextract according to a known method using alcohol precipitation.

As the glycosaminoglycans and salts thereof, commercially availablehyaluronic acid, chondroitin, chondroitin sulfate, chondroitinhydrochloride, keratan sulfate, heparin, heparan sulfate and dermatansulfate or salts thereof may also be used.

The pharmaceuticals, the foods and drinks, the animal feeds, the foodadditives and the feed additives of the present invention, and themethod for preventing or treating arthritis in humans or non-humananimals using them are described below.

The present invention can be applied to any arthritis includingchlamydial arthritis, chronic absorptive arthritis, enteropathicarthritis, gonococcal arthritis, gouty arthritis, Jaccoud arthritis,juvenile arthritis, Lyme arthritis, ochronotic arthritis, suppurativearthritis, osteoarthritis, periarthritis scapulohumeralis (so-calledfrozen shoulder), arthritis caused by excessive work load and rheumatoidarthritis, and is particularly advantageous to rheumatoid arthritis.

-   (a) a Pharmaceutical comprising N-acylated hydroxyproline derivative    or salt thereof, and amino sugar or salt thereof and/or    glycosaminoglycan or salt thereof as active ingredients, and method    for preventing or treating arthritis in humans or non-human animals    using the pharmaceutical

The pharmaceutical of the present invention includes any pharmaceuticalsthat comprise the N-acylated hydroxyproline derivative or a saltthereof, and the amino sugar or a salt thereof and/or theglycosaminoglycan or a salt thereof as active ingredients. They arepreferably used as an arthritis preventing or treating agent.

In addition to the N-acylated hydroxyproline derivative or a saltthereof, and the amino sugar or a salt thereof and/or theglycosaminoglycan or a salt thereof, the pharmaceutical of the presentinvention may also comprise other optional ingredients that areeffective in preventing or treating arthritis.

Examples of the other ingredients that are effective in preventing ortreating arthritis (hereinafter also referred to simply as “other activeingredients”) are purified products or extracts of, or productscontaining boron, calcium, chromium, copper, magnesium, manganese,selenium, silicone, zinc, S-adenosyl methionine, collagen, collagenhydrolysate, gelatin, gelatin hydrolysate, bromelain, trypsin,chymotrypsin, papain, rutin, carotenoid, flavonoid, antioxidantvitamins, γ-linolenic acid, eicosapentaenoic acid, boswellia, capsaicin,cat's claw, devil's claw, fever few, ginger, nettles, niacinamide, sharkcartilage, turmeric, curcumin, and the like.

The pharmaceutical of the present invention is produced by optionalmethods well known in the technical field of pharmaceutics by mixing theN-acylated hydroxyproline derivative or a salt thereof, and the aminosugar or a salt thereof and/or the glycosaminoglycan or a salt thereofas well as other active ingredients as required together with one ormore pharmaceutically acceptable carriers.

In administering the pharmaceutical of the present invention, it isdesirable to select a route of administration that is effective in theprevention or the treatment of arthritis, examples thereof being oraladministration and parenteral administrations such as intravenousadministration.

Examples of the dosage form are tablets, capsules, injections, drops,syrups, sublingual tablets, various types of creams and suppositories.

Liquid preparations such as syrups that are suitable for oraladministration can be produced using water, sugars such as sucrose,sorbitol and fructose, glycols such as polyethylene glycol and propyleneglycol, oils such as sesame oil, olive oil and soybean oil, antisepticssuch as p-hydroxybenzoic acid esters, flavors such as strawberry flavorand peppermint, etc. Furthermore, tablets, powders and granules can beproduced using excipients such as lactose, glucose, sucrose andmannitol, disintegrators such as starch and sodium alginate, lubricantssuch as magnesium stearate and talc, binders such as polyvinyl alcohol,hydroxypropyl cellulose and gelatin, surfactants such as fatty acidesters, plasticizers such as glycerin, etc.

Preparations appropriate for parenteral administration comprise,preferably, a sterilized aqueous agent containing an active compound,which is isotonic to the recipient's blood. In the case of an injection,for example, an injectable solution is prepared using a carrierconsisting of a salt solution, a glucose solution or a mixture of salineand a glucose solution.

In producing these parenteral preparations, it is also possible to addone or more supplementary components selected from diluents,antiseptics, flavors, excipients, disintegrators, lubricants, binders,surfactants, plasticizers and the like exemplified in connection withoral preparations.

The content of the N-acylated hydroxyploline derivative or a saltthereof in the pharmaceutical of the present invention is 1–1000 mg/g,preferably 10–500 mg/g, especially preferably 100–200 mg/g of thepharmaceutical.

The content of the amino sugar or a salt thereof and theglycosaminoglycan or a salt thereof in the pharmaceutical of the presentinvention is not restricted so far as at least either of them iscontained in an amount of 1–1000 mg/g, preferably 10–500 mg/g,especially preferably 100–200 mg/g of the pharmaceutical.

The composition ratio of the N-acylated hydroxyproline derivative or asalt thereof to the amino sugar or a salt thereof and/or theglycosaminoglycan or a salt thereof in the pharmaceutical of the presentinvention is 1:100–100:1, preferably 1:50–50:1, especially preferably10:1–1:10 in terms of weight ratio.

The dosage and the frequency of administration of the pharmaceutical ofthe present invention vary depending on the mode of administration, theage, the body weight and the symptoms of the patient.

In the case of oral administration, the pharmaceutical is administeredin a dose of 1–5000 mg, preferably 10–1000 mg, especially preferably100–500 mg as N-acylated hydroxyproline derivative or salt thereof andamino sugar or salt thereof and/or glycosaminoglycan or salt thereof,respectively, per adult person once to several times a day.

In the case of parenteral administration such as intravenousadministration, the pharmaceutical is administered in a dose of 0.5–5000mg, preferably 5–1000 mg, especially preferably 50–500 mg as N-acylatedhydroxyproline derivative or salt thereof, and amino sugar or saltthereof and/or glycosaminoglycan or salt thereof, respectively, peradult person once to several times a day.

By administering the pharmaceutical of the present invention on a dailybasis, it is possible to prevent arthritis.

When arthritis has already been developed, it is possible to treat it byadministering the pharmaceutical on a daily basis. The dosing period isusually one week to 10 years, preferably one month to 5 years.

The term “to prevent arthritis” as used herein means bringing about aneffect of completely preventing the development of arthritis, reducingthe incidence or suppressing the symptoms at the time of the onset byingesting the foods and drinks, animal feeds, food additives or feedadditives to be explained in (b) below or the above-describedpharmaceutical on a daily basis.

On the other hand, the term “to treat arthritis” as used herein meansbringing about an effect of relieving or treating the symptoms byadministering the above-described pharmaceutical after the onset ofarthritis.

The pharmaceutical of the present invention can be used not only forhumans but also for animals other than humans (non-human animals). Whenused for non-human animals, the dose is 0.02–100 mg/kg, preferably0.2–20 mg/kg, especially preferably 2–10 mg/kg of the body weight of thenon-human animal to which the pharmaceutical is administered asN-acylated hydroxyproline derivative or salt thereof, and amino sugar orsalt thereof and/or glycosaminoglycan or salt thereof, respectively.

The active ingredients of the above pharmaceutical are not necessarilyadministered simultaneously so far as they are administered within theperiod for which each of them has an effect.

Furthermore, the above pharmaceutical may be prepared so that theN-acylated hydroxyproline derivative or a salt thereof, and the aminosugar or a salt thereof and/or the glycosaminoglycan or a salt thereofare contained in the same preparation. It may also be prepared as apreparation in the form of a kit.

The preparation in the form of a kit (hereinafter referred to simply as“a kit”) as used herein means two or more preparations preparedaccording to a conventional method by mixing a substance or acombination of substances selected from the N-acylated hydroxyprolinederivatives or salts thereof, the amino sugars or salts thereof and theglycosaminoglycans or salts thereof with one or more pharmaceuticallyacceptable carriers, any of which comprises the N-acylatedhydroxyproline derivative or a salt thereof as an active ingredient.

Examples of the combination of preparations constituting the kit are acombination of a preparation comprising the N-acylated hydroxyprolinederivative or a salt thereof and a preparation comprising the aminosugar or a salt thereof; a combination of a preparation comprising theN-acylated hydroxyproline derivative or a salt thereof and a preparationcomprising the glycosaminoglycan or a salt thereof; a combination of apreparation comprising the N-acylated hydroxyproline derivative or asalt thereof, a preparation comprising the amino sugar or a salt thereofand a preparation comprising the glycosaminoglycan or a salt thereof;and a combination of a preparation comprising the N-acylatedhydroxyproline derivative or a salt thereof and a preparation comprisingthe amino sugar or a salt thereof and the glycosaminoglycan or a saltthereof, although the combination is not limited thereto.

Each of the preparations included in the kit may be in any form so longas they exist separately. For example, they may be packed separately ormay be present as a mixture in the same vial.

In administering the preparations in the form of a kit, they may beadministered either simultaneously or separately.

When administered separately, it is desirable that the preparations areadministered within the period during which the active ingredientscontained in the preparations are highly effective in a body. Forexample, all preparations are administered within 8 hours, preferablywithin 2 hours per one administration.

For the kit-form preparations, the dose is set so that the total doseper day of each active ingredient in the preparations corresponds to theabove-described daily dose of each ingredient.

-   (b) Foods and drinks, animal feeds, food additives and feed    additives comprising N-acylated hydroxyproline derivative or a salt    thereof, and amino sugar or a salt thereof and/or glycosaminoglycan    or a salt thereof, and method for preventing or treating arthritis    in humans or non-human animals using them

The foods and drinks of the present invention are obtained by adding theN-acylated hydroxyproline derivative or a salt thereof, and the aminosugar or a salt thereof and/or the glycosaminoglycan or a salt thereofto foods and drinks.

The foods and drinks of the present invention also include thoseobtained by the addition of the food additives of the present invention.

The foods and drinks of the present invention further include thoseobtained by adding other active ingredients described in (a) above inaddition to the N-acylated hydroxyproline derivative or a salt thereof,and the amino sugar or a salt thereof and/or the glycosaminoglycan or asalt thereof.

Except the addition of the N-acylated hydroxyproline derivative or asalt thereof, and the amino sugar or a salt thereof and/or theglycosaminoglycan or a salt thereof, or other active ingredients asrequired, the foods and drinks of the present invention can be producedusing a process generally used for producing foods and drinks.

The foods and drinks of the present invention may be in any of the formsincluding juice, soft drinks, tea, lactic acid beverages, fermentedmilk, ices, dairy products such as butter, cheese, yogurt, processedmilk and skim milk, meat products such as ham, sausages and hamburger,fish products such as steamed, baked or fried fish paste, egg productssuch as baked or steamed foods made of beaten eggs, confectionery suchas cookies, jellies, chewing gum, candies and snacks, bread, noodles,pickles, smoked foods, dried fish, preserved foods boiled down in soysauce, salted foods, soups and seasonings.

Furthermore, the foods and drinks of the present invention may take theform of a powdered food, a sheet-shaped food, a bottled food, a cannedfood, a retort food, a capsule food, a tablet food, a liquid food, ahealth drink, etc.

The foods and drinks of the present invention can be used as a healthfood or a functional food having an effect on the prevention ortreatment of arthritis.

When the foods and drinks of the present invention are a drink or atablet, for example, they can be prepared by adding, to the N-acylatedhydroxyproline derivative or a salt thereof, and the amino sugar or asalt thereof and/or the glycosaminoglycan or a salt thereof, otheractive ingredients, additives, etc. as required and then dissolving ordispersing the mixture in an appropriate amount of water or tabletingthe mixture. Furthermore, when the foods and drinks of the presentinvention are confectionary such as caramels, drops, chocolate, jelly,biscuits and cookies, they can be prepared by adding, to the N-acylatedhydroxyproline derivative or a salt thereof, and the amino sugar or asalt thereof and/or the glycosaminoglycan or a salt thereof, otheractive ingredients, additives, etc. as required and additionallyappropriate carriers such as wheat flour, rice flour, starch, cornstarch, soybean, etc. as required and shaping the obtained mixture intoan appropriate form according to a conventional method.

The foods and drinks of the present invention can also be produced byusing granulating methods such as fluidized bed granulation, stirringgranulation, extrusion granulation, rolling granulation, air streamgranulation, compression molding granulation, disruption granulation,spray granulation and blasting granulation, coating methods such as pancoating, fluidized bed coating and dry coating, plumping methods such aspuff drying, excess steam method, foam mat method and microwave heatingmethod, and extrusion methods using an extruding granulator or anextruder.

The food additives of the present invention can be prepared according tomethods similar to those mentioned in (a) above with respect to oralpreparations. They can be produced, for example, into powder, granules,pellets, tablets and various liquid preparations by mixing with ordissolving together with other food additives as required.

To the foods and drinks or food additives of the present invention maybe added food additives generally employed in foods and drinks such assweeteners, coloring agents, preservatives, thickening stabilizers,antioxidants, color developing agents, bleaching agents, fungicides, gumbases, bittering agents, enzymes, glazing agents, acidulants,seasonings, emulsifiers, nutrient supplements, additional materials forpreparation, flavors and spice extracts.

To the foods and drinks of the present invention, the N-acylatedhydroxyproline derivative or a salt thereof, and the amino sugar or asalt thereof and/or the glycosaminoglycan or a salt thereof are added sothat they are ingested generally in an amount of 1–5000 mg, preferably10–1000 mg, especially preferably 100–500 mg per adult person per day,respectively, although the amount may vary depending upon the form ofthe foods and drinks.

These foods and drinks may be ingested once or in several divided partsa day.

The composition ratio of the N-acylated hydroxyproline derivative or asalt thereof to the amino sugar or a salt thereof and/or theglycosaminoglycan or a salt thereof in the foods and drinks of thepresent invention is 1:100–100:1, preferably 1:50–50:1, especiallypreferably 10:1–1:10 in terms of weight ratio.

The period of ingestion is usually one week to 10 years, preferably onemonth to 5 years.

As in the case of the pharmaceutical of the present invention, the foodsand drinks of the present invention may be produced so that theN-acylated hydroxyproline derivative or a salt thereof, and the aminosugar or a salt thereof and/or the glycosaminoglycan or a salt thereofare contained in the same food and drink, or may be prepared as a set offoods and drinks.

Examples of the combination of the set of foods and drinks are acombination of a food and drink comprising the N-acylated hydroxyprolinederivative or a salt thereof and a food and drink comprising the aminosugar or a salt thereof; a combination of a food and drink comprisingthe N-acylated hydroxyproline derivative or a salt thereof and a foodand drink comprising the glycosaminoglycan or a salt thereof; acombination of a food and drink comprising the N-acylated hydroxyprolinederivative or a salt thereof, a food and drink comprising the aminosugar or a salt thereof and a food and drink comprising theglycosaminoglycan or a salt thereof; and a combination of a food anddrink comprising the N-acylated hydroxyproline derivative or a saltthereof and a food and drink comprising the amino sugar or a saltthereof and the glycosaminoglycan or a salt thereof, although thecombination is not limited thereto.

Each of the foods and drinks included in the set may be in any form solong as they exist separately. For example, they may be packedseparately or may be present as a mixture in the same container.

In ingesting the set of the foods and drinks, they may be ingestedeither simultaneously or separately.

When separately ingested, it is desirable that the foods and drinks areingested within the period during which the active ingredients containedin the foods and drinks are highly effective in a body. For example, allfoods and drinks are ingested within 8 hours, preferably within 2 hoursper one intake.

The intake of the foods and drinks is set so that the total intake perday of each active ingredient in the foods and drinks corresponds to theabove-described daily intake of each ingredient.

The animal feeds of the present invention comprise an animal feed towhich the N-acylated hydroxyproline derivative or a salt thereof, andthe amino sugar or a salt thereof and/or the glycosaminoglycan or a saltthereof are added. If necessary, the other active ingredients describedin (a) above may also be added thereto.

The animal feeds of the present invention include any feeds that preventor treat arthritis in non-human animals, preferably vertebrates, such asmammals other than humans, birds, reptiles, amphibians and fish,examples thereof being feeds for pets such as dogs, cats, rabbits andmice, pet food, feeds for livestock such as cows and pigs, feeds forpoultry such as hens and turkeys, feeds for reptiles such as lizards,crocodiles and iguanas, feeds for amphibians such as frogs and newts andfeeds for cultivated fish such as sea breams and young yellowtails.

The animal feeds of the present invention can be used as healthsupplement feeds for animals having an effect on the prevention or thetreatment of arthritis.

Except the addition of the N-acylated hydroxyproline derivative or asalt thereof, and the amino sugar or a salt thereof and/or theglycosaminoglycan or a salt thereof, and additionally other activeingredients, if necessary, or the feed additive of the present inventionto a feed, the animal feeds of the present invention can be producedusing a process generally used for producing feeds.

The feeds include grains, bran, vegetable oil cakes, animal-based feed,other feeds and purified products, or mixtures thereof.

Examples of the grains are milo, wheat, barley, oats, rye, brown rice,buckwheat, foxtail millet, broomcorn millet, Japanese millet, corn andsoybean.

Examples of the bran are rice bran, defatted rice bran, wheat bran,wheat middlings, wheat, germ, barley bran, pellet, corn bran and corngerm.

Examples of the vegetable oil cakes are soybean oil cake, soybean flour,linseed oil cake, cottonseed oil cake, peanut oil cake, safflower oilcake, coconut oil cake, palm oil cake, sesame oil cake, sunflower oilcake, rapeseed oil cake, kapok oil cake and mustard seed oil cake.

Examples of the animal-based feed are fish meal such as northern oceanmeal, imported meal, whole meal and coastal meal, fish soluble, meatmeal, meat and bone meal, blood powder, degradated hair, bone meal,treated by-products for livestock, feather meal, silkworm pupa, skimmilk, casein and dry whey.

Examples of other feeds are stalks and leaves of plants such as alfalfa,hay cube, alfalfa leaf meal and powder of false acacia, by-products fromthe corn processing industry such as corn gluten, meal, corn gluten feedand corn steep liquor, processed starch products such as starch,products from the fermentation industry such as yeast, beer cake, maltroot, alcohol cake and soy sauce cake, agricultural by-products such asprocessed citrus fruit cake, tofu cake, coffee cake and cocoa cake,cassava, broad bean, guar meal, seaweeds, krill, spirulina, chlorellaand minerals.

Examples of the purified products are proteins such as casein andalbumin, amino acids, sugars such as starch, cellulose, sucrose andglucose, minerals and vitamins.

The animal feeds of the present invention can also be produced by usinggranulating methods such as fluidized bed granulation, stirringgranulation, extrusion granulation, rolling granulation, air streamgranulation, compression molding granulation, disruption granulation,spray granulation and blasting granulation, coating methods such as pancoating, fluidized bed coating and dry coating, plumping methods such aspuff drying, excess steam method, foam mat method and microwave heatingmethod and extrusion methods using an extruding granulator or anextruder.

The feed additives of the present invention can be prepared according tomethods similar to those mentioned in (a) above with respect to oralpreparations. They can be produced, for example, into powder, granules,pellets, tables and various liquid preparations generally by mixing withor dissolving together with other feed additives as required.

The N-acylated hydroxyproline derivative or a salt thereof, and theamino sugar or a salt thereof and/or the glycosaminoglycan or a saltthereof in the animal feeds of the present invention are added so as tobe ingested generally in an amount of 0.02–100 mg/kg, preferably 0.2–20mg/kg, especially preferably 2–10 mg/kg although the amount variesdepending upon the form of intake, the kinds of non-human animals toingest them and the age and the body weight of the non-human animals.

The animal feeds are fed once or in several parts a day. It is alsopossible to administer the feed additives of the present invention as anoral preparation for the prevention or the treatment of arthritis fornon-human animals in the same dose and for the same period as in thecase of the feeds described above.

There is no restriction as to the period for which the feeds are to begiven, as it varies depending upon the non-human animal to ingest them.Usually, the period is one week to 5 years, preferably 2 weeks to 2years.

As in the case of the pharmaceutical of the present invention, theanimal feeds of the present invention may be produced so that theN-acylated hydroxyproline derivative or a salt thereof, and the aminosugar or a salt thereof and/or the glycosaminoglycan or a salt thereofare contained in the same animal feed. They may also be produced as aset of feeds.

Examples of the combination of the set of feeds are a combination of ananimal feed comprising the N-acylated hydroxyproline derivative or asalt thereof and an animal feed comprising the amino sugar or a saltthereof; a combination of an animal feed comprising the N-acylatedhydroxyproline derivative or a salt thereof and an animal feedcomprising the glycosaminoglycan or a salt thereof; a combination of ananimal feed comprising the N-acylated hydroxyproline derivative or asalt thereof, an animal feed comprising the amino sugar or a saltthereof and an animal feed comprising the glycosaminoglycan or a saltthereof; and a combination of an animal feed comprising the N-acylatedhydroxyproline derivative or a salt thereof and an animal feedcomprising the amino sugar or a salt thereof and the glycosaminoglycanor a salt thereof, although the combination is not limited thereto.

Each of the animal feeds included in the set may be in any form so longas they exist separately. For example, they may be packed separately ormay be present as a mixture in the same container.

In feeding the set of the animal feeds, the feeds may each be fed eithersimultaneously or separately.

When fed separately, it is desirable that the animal feeds are ingestedwithin the period during which the active ingredients contained in theanimal feeds are highly effective in a body. For example, animal feedsare ingested within 8 hours, preferably within 2 hours per one intake.

For the set of animal feeds, the intake is set so that the total intakeper day of each active ingredient in the animal feeds corresponds to theabove-described daily intake of each ingredient.

Test examples of the present invention are shown below.

Unless otherwise noted, in the following test examples, DBA/1J miceproduced by Charles River, powder feed CE2 produced by CLEA Japan,N-acetylhydroxyproline produced by Kyowa Hakko Kogyo, D-glucosaminesulfate 2NaCl as glucosamine (Miyako Kagaku) and chondroitin sodiumsulfate (Maruha Kagaku) as chondtoitin were used.

The amounts of the additives in the examples are all shown by weight %.

TEST EXAMPLE 1

Effect of N-acetylhydroxyproline and Glucosamine in Mice with Type IICollagen-Induced Arthritis

It is known that arthritis is induced in DBA/1J mice by administeringtype II collagen two times.

As the first administration of type II collagen, a solution prepared bymixing an equal amount of type II collagen [Collagen Gijutsu KenshuKaisha (MCK)] and Freund's complete adjuvant (Iatron) and emulsifyingthe mixture using a homogenizer was intradermally administered to 6-weekold male DBA/1J mice in an amount of 100 μl per one animal.

Twenty-one days after the first administration of type II collagen,EXAMPLE a solution prepared by mixing an equal amount of type IIcollagen and Freund's complete adjuvant and emulsifying the mixtureusing a homogenizer in a similar manner was intradermally administeredto the animals in an amount of 100 μl per one animal as the secondadministration of type II collagen. In this manner, arthritis wasinduced in mice.

Starting on the day of the first administration of type II collagen, themice were given powder feed CE-2 containing no additive as control;powder feed CE-2 containing 0.1% N-acetylhydroxyproline (indicated asAcHYP in Tables 1-1 and 1-2); powder feed CE-2 containing 0.1%glucosamine (D-glucosamine sulfate 2NaCl); and powder feed CE-2containing 0.05% N-acetylhydroxyproline and 0.05% glucosamine,respectively. At days 24, 28, 31, 34, 38 and 42 after the firstadministration of type II collagen, the extent of the development ofarthritis was scored according to the following indices.

Scoring was carried out by applying 0–4 points with respect to one offour paws of mice: 0: no symptom; 1: swelling of one finger or swelling(slight) of the ankle; 2: swelling of 1–3 fingers or swelling of theankle; 3: swelling of 3–5 fingers plus swelling of the ankle; and 4:swelling of all the fingers plus swelling of the ankle. Each mouse wasscored the total of the points of 4 paws, namely, 0–16 points.

Twenty mice were subjected to the test with respect to each of theconditions.

The results are shown in Tables 1-1 and 1-2. Table 1-2 is a continuationto Table 1-1.

In the tables, the figures show the scores under different treatmentconditions, which are given as mean±SE (N=20).

In the tables, “no treatment” means no administration of type IIcollagen, and “control” means mice were given the feed containing noadditive.

TABLE 1-1 Score of arthritis under various treatment conditions Timecourse (days) Treatment 0 24 28 31 No treatment 0.0 ± 0.0 0.0 ± 0.0 0.0± 0.0  0.0 ± 0.0 Control 0.0 ± 0.0 0.1 ± 0.1 6.8 ± 0.5 10.0 ± 0.6Glucosamine  0.1% 0.0 ± 0.0 0.1 ± 0.1 5.8 ± 0.6  7.7 ± 0.7 AcHYP  0.1%0.0 ± 0.0 0.0 ± 0.0 6.5 ± 0.7  9.5 ± 0.8 AcHYP + 0.05% 0.0 ± 0.0 0.0 ±0.0 4.2 ± 0.7  6.9 ± 0.9 Glucosamine 0.05% AcHYP: N-acetylhydroxyproline

TABLE 1-2 Score of arthritis under various treatment conditions(Continued from Table 1-1) Time course (days) Treatment 34 38 42 Notreatment  0.0 ± 0.0  0.0 ± 0.0  0.0 ± 0.0 Control 11.0 ± 0.6 11.0 ± 0.610.8 ± 0.6 Glucosamine  0.1%  9.2 ± 0.7  9.5 ± 0.6  9.4 ± 0.6 AcHYP 0.1% 10.7 ± 0.8 10.8 ± 0.8 10.6 ± 0.8 AcHYP + 0.05%  8.1 ± 1.0  8.5 ±0.9  8.5 ± 1.0 Glucosamine 0.05% AcHYP: N-acetylhydroxyproline

As shown in Tables 1-1 and 1-2, lowering of the scores was observed inthe case where 0.1% glucosamine or 0.1% N-acetylhydroxyproline was addedto the feed compared with the case where no additive was added to thefeed (control).

Furthermore, prominent lowering of the scores was observed in the casewhere 0.05% each glucosamine and N-acetylhydroxyproline are added to thefeed compared with the case where 0.1% glucosamine or 0.1%N-acetylhydroxyproline was added.

TEST EXAMPLE 2

Effect of N-acetylhydroxyproline and Chondroitin in Mice with Type IICollagen-Induced Arthritis

An experiment similar to that of Test Example 1 was carried out exceptthat chondroitin was used in place of glucosamine.

That is, 21 days after the first administration of type II collagen, asolution prepared by mixing an equal amount of type II collagen andFreund's complete adjuvant and emulsifying the mixture using ahomogenizer in a similar manner as in Test Example 1 was intradermallyadministered to the mice in an amount of 100 μl per one animal as thesecond administration of type II collagen. In this manner, arthritis wasinduced in mice.

Starting on the day of the first administration of type II collagen, themice were given powder feed CE-2 containing no additive as control;powder feed CE-2 containing 0.1% N-acetylhydroxyproline (indicated asAcHYP in Tables 2-1 and 2-2); powder feed CE-2 containing 0.1%chondroitin; and powder feed CE-2 containing 0.05%N-acetylhydroxyproline and 0.05% chondroitin, respectively. At days 23,27, 30, 33, 36, 40 and 42 after the first administration of type IIcollagen, the extent of the development of arthritis was scoredaccording to the indices employed in the above Test Example 1.

Twenty mice were subjected to the test with respect to each of theconditions.

The results are shown in Tables 2-1 and 2-2. Table 2-2 is a continuationto Table 2-1. In the tables, the figures show the scores under differenttreatment conditions, which are given as mean±SE (N=20).

In the tables, “no treatment” means no administration of type IIcollagen, and “control” means mice were given the feed containing noadditive.

TABLE 2-1 Score of arthritis under various treatment conditions Timecourse (days) Treatment 0 23 27 30 No treatment 0.0 ± 0.0 0.0 ± 0.0 0.0± 0.0 0.0 ± 0.0 Control 0.0 ± 0.0 0.0 ± 0.0 3.8 ± 0.5 8.9 ± 0.7Chondroitin  0.1% 0.0 ± 0.0 0.1 ± 0.1 3.4 ± 0.6 7.8 ± 1.0 AcHYP  0.1%0.0 ± 0.0 0.1 ± 0.1 3.3 ± 0.7 7.0 ± 0.9 AcHYP + 0.05% 0.0 ± 0.0 0.1 ±0.1 2.6 ± 0.5 5.9 ± 1.0 Chondroitin 0.05% AcHYP: N-acetylhydroxyproline

TABLE 2-2 Score of arthritis under various treatment conditions(Continued from Table 2-1) Time course (days) Treatment 33 36 40 42 Notreatment 0.0 ± 0.0 0.0 ± 0.0  0.0 ± 0.0  0.0 ± 0.0 Control 9.8 ± 0.89.8 ± 0.8 10.0 ± 0.8 10.0 ± 0.8 Chondroitin  0.1% 9.1 ± 0.9 9.7 ± 0.8 9.9 ± 0.8  9.8 ± 0.7 AcHYP  0.1% 7.8 ± 0.9 7.9 ± 1.0  8.4 ± 0.9  8.8 ±0.8 AcHYP + 0.05% 7.4 ± 1.0 7.8 ± 0.9  7.9 ± 1.0  7.9 ± 1.0 Chondroitin0.05% AcHYP: N-acetylhydroxyproline

As shown in Tables 2-1 and 2-2, in the case where 0.1%N-acetylhydroxyproline was added, lowering of the score was observed atany of the days after day 27 compared with the case where no additivewas added to the feed (control).

In the case where 0.1% chondroitin was added to the feed, slightlowering of the score compared with control was observed until day 33,but almost no lowering of the score was observed after day 36.

On the other hand, in the case where 0.05% each chondroitin andN-acetylhydroxyproline were added to the feed, lowering of the scorecompared with control was observed at any of the days after day 27. Thelowering of the score was prominent compared with the cases where 0.1%chondroitin or 0.1% N-acetylhydroxyproline was added alone.

TEST EXAMPLE 3

Effect of N-acetylhydroxyproline, Glucosamine and Chondroitin in Micewith Type II Collagen-Induced Arthritis

An experiment similar to that of Test Example 1 was carried out exceptthat glucosamine and chondroitin were used in place of glucosamine.

That is, 21 days after the first administration of type II collagen, asolution prepared by mixing an equal amount of type II collagen andFreund's complete adjuvant and emulsifying the mixture using ahomogenizer in a similar manner as in Test Example 1 was intradermallyadministered to the mice in an amount of 100 μl per one animal as thesecond administration of type II collagen. In this manner, arthritis wasinduced in mice.

Starting on the day of the first administration of type II collagen, themice were given powder feed CE-2 containing no additive as control;powder feed CE-2 containing 0.05% N-acetylhydroxyproline (indicated asAcHYP in Tables 3-1 and 3-2); powder feed CE-2 containing 0.05%N-acetylhydroxyproline and 0.05% chondroitin; and powder feed CE-2containing 0.05% glucosamine, 0.05% glucosamine and 0.05% chondroitin,respectively. At days 24, 28, 31, 35, 38 and 42 after the firstadministration of type II collagen, the extent of the development ofarthritis was scored according to the indices employed in the above TestExample 1.

Twenty mice were subjected to the test with respect to each of theconditions.

The results are shown in Tables 3-1 and 3-2. Table 3-2 is a continuationto Table 3-1. In the tables, the figures show the scores under differenttreatment conditions, which are given as mean±SE (N=20).

In the tables, “no treatment” means no administration of type IIcollagen, and “control” means mice were given the feed containing noadditive.

TABLE 3-1 Score of arthritis under various treatment conditions Timecourse (days) Treatment 0 24 28 31 No treatment 0.0 ± 0.0 0.0 ± 0.0 0.0± 0.0 0.0 ± 0.0 Control 0.0 ± 0.0 0.2 ± 0.1 5.7 ± 0.6 9.5 ± 0.7 AcHYP0.05% 0.0 ± 0.0 0.2 ± 0.1 5.4 ± 0.6 8.5 ± 0.7 Chondroitin + 0.05% 0.0 ±0.0 0.2 ± 0.1 4.5 ± 0.7 8.4 ± 0.8 Glucosamine 0.05% AcHYP + 0.05% 0.0 ±0.0 0.1 ± 0.1 4.1 ± 0.5 7.3 ± 0.8 Glucosamine + 0.05% Chondroitin 0.05%AcHYP: N-acetylhydroxyproline

TABLE 3-2 Score of arthritis under various treatment conditions(Continued from Table 3-1) Time course (days) Treatment 35 38 42 Notreatment 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 Control 9.7 ± 0.7 9.8 ± 0.7 10.2± 0.7  AcHYP 0.05% 9.4 ± 0.8 9.9 ± 0.8 9.4 ± 0.7 Chondroitin + 0.05% 9.4± 0.8 9.7 ± 0.7 9.5 ± 0.8 Glucosamine 0.05% AcHYP + 0.05% 8.4 ± 0.8 8.4± 0.8 8.5 ± 0.8 Glucosamine + 0.05% Chondroitin 0.05% AcHYP:N-acetylhydroxyproline

As shown in Tables 3-1 and 3-2, in the cases where 0.05%N-acetylhydroxyproline was added and where 0.05% each glucosamine andchondroitin were added to the feed, almost no lowering of the scores wasobserved at any of the days after day 24 compared with the case where noadditive was added to the feed (control).

On the other hand, where 0.05% each glucosamine, chondroitin andN-acetylhydroxyproline were added to the feed, lowering of the scorescompared with control was observed at any of the days after day 24.

TEST EXAMPLE 4

Effect of N-acetylhydroxyproline and Glucosamine Plus Chondroitin inMice with Type II Collagen-Induced Arthritis

As in Test Example 1, 21 days after the first administration of type IIcollagen, a solution prepared by mixing an equal amount of type IIcollagen and Freund's complete adjuvant and emulsifying the mixtureusing a homogenizer in a similar manner as in Test Example 1 wasintradermally administered to the mice in an amount of 100 μl per oneanimal as the second administration of type II collagen. In this manner,arthritis was induced in mice.

Starting at day 28 after the first administration of type II collagen,the mice were given powder feed CE-2 containing no additive as control;powder feed CE-2 containing 0.1% N-acetylhydroxyproline (indicated asAcHYP in Tables 4-1 and 4-2); powder feed CE-2 containing 0.1%glucosamine; powder feed CE-2 containing 0.05% N-acetylhydroxyprolineand 0.05% glucosamine; powder feed CE-2 containing 0.05% glucosamine and0.05% chondroitin; and powder feed CE-2 containing 0.05%N-acetylhydroxyproline, 0.025% glucosamine and 0.025% chondroitin,respectively. At days 28, 33, 36, 39 and 42 after the firstadministration of type II collagen, the extent of the development ofarthritis was scored according to the indices employed in the above TestExample 1.

Ten mice were subjected to the test with respect to each of theconditions.

The results are shown in Tables 4-1 and 4-2. Table 4-2 is a continuationto Table 4-1. In the tables, the figures show the scores under differenttreatment conditions, which are given as mean±SE (N=10).

In the tables, “no treatment” means no administration of type IIcollagen, and “control” means mice were given the feed containing noadditive.

TABLE 4-1 Score of arthritis under various treatment conditions Timecourse (days) Treatment 0 28 33 No treatment 0.0 ± 0.0 0.0 ± 0.0 0.0 ±0.0 Control 0.0 ± 0.0 4.5 ± 1.0 11.0 ± 1.3  AcHYP  0.1% 0.0 ± 0.0 4.3 ±0.9 9.8 ± 1.0 Glucosamine  0.1% 0.0 ± 0.0 4.3 ± 0.9 9.8 ± 1.2 AcHYP + 0.05% 0.0 ± 0.0 4.2 ± 0.7 8.7 ± 1.0 Glucosamine  0.05% Glucosamine + 0.05% 0.0 ± 0.0 4.3 ± 1.3 9.3 ± 1.3 Chondroitin  0.05% AcHYP +  0.05%0.0 ± 0.0 4.0 ± 0.9 8.7 ± 1.2 Glucosamine + 0.025% Chondroitin 0.025%AcHYP: N-acetylhydroxyproline

TABLE 4-2 Score of arthritis under various treatment conditions(Continued from Table 4-1) Time course (days) Treatment 36 39 42 Notreatment  0.0 ± 0.0  0.0 ± 0.0  0.0 ± 0.0 Control 11.4 ± 1.4 11.8 ± 1.311.5 ± 1.4 AcHYP  0.1% 10.9 ± 1.1 10.7 ± 1.1 10.8 ± 1.0 Glucosamine 0.1% 10.1 ± 1.1 10.4 ± 1.0 10.6 ± 1.0 AcHYP +  0.05% 10.0 ± 1.0  9.5 ±0.9  9.9 ± 1.0 Glucosamine  0.05% Glucosamine +  0.05% 10.0 ± 1.4 10.9 ±1.3 10.7 ± 1.2 Chondroitin  0.05% AcHYP +  0.05%  9.2 ± 1.4  9.4 ± 1.4 9.7 ± 1.3 Glucosamine + 0.025% Chondroitin 0.025% AcHYP:N-acetylhydroxyproline

As shown in Tables 4-1 and 4-2, in the cases where 0.1%N-acetylhydroxyproline was added and where 0.1% glucosamine was added tothe feed, slight lowering of the scores compared with control wasobserved at any of the days after day 33.

In the case where 0.05% each glucoseamine and chondroitin were added tothe feed, slight lowering of the scores compared with control wasobserved at any of the days after day 33.

On the other hand, in the cases where 0.05% N-acetylhydroxyproline and0.05% glucosamine were added to the feed and where 0.05%N-acetylhydroxyproline, 0.025% glucosamine and 0.025% chondroitin wereadded to the feed, lowering of the scores was prominent compared withcontrol at any of the days after day 33.

Examples of the present invention are shown below.

Best Modes for Carrying out the Invetion

Unless otherwise noted, in the following examples,N-acetylhydroxyproline produced by Kyowa Hakko Kogyo, D-glucosaminesulfate 2NaCl as glucosamine (Miyako kagaku) and chondroitin sodiumsulfate (Maruha Kagaku) as chondtoitin were used.

EXAMPLE 1

Tablets of 8 mm in diameter and 200 mg in weight each are prepared bymixing the ingredients according to the composition shown in Table 5below and tableting the resulting mixture using a tableting machine(Hata Seisakusho, HT-AP15SS-U).

TABLE 5 Composition Mixing rate (wt %) N-Acetylhydroxyproline 20Glucosamine 10 Lactose 30 Calcium lactate 10 Magnesium stearate 25Calcium carbonate  5

EXAMPLE 2

Tablets of 8 mm in diameter and 200 mg in weight each were prepared bymixing the ingredients according to the composition shown in Table 6below and tableting the resulting mixture using a tableting machine(Hata Seisakusho, HT-AP15SS-U).

TABLE 6 Composition Mixing rate (wt %) N-Acetylhydroxyproline 20Glucosamine 20 Lactose 20 Calcium lactate 10 Magnesium stearate 25Calcium carbonate  5

EXAMPLE 3

Tablets of 8 mm in diameter and 200 mg in weight each were prepared bymixing the ingredients according to the composition shown in Table 7below and tableting the resulting mixture using a tableting machine(Hata Seisakusho, HT-AP15SS-U).

TABLE 7 Composition Mixing rate (wt %) N-Acetylhydroxyproline 15Glucosamine 15 Chondroitin 15 Lactose 15 Calcium lactate 10 Magnesiumstearate 25 Calcium carbonate  5

EXAMPLE 4

Tablets of 8 mm in diameter and 200 mg in weight each were prepared bymixing the ingredients according to the composition shown in Table 8below and tableting the resulting mixture using a tableting machine(Hata Seisakusho, HT-AP15SS-U).

TABLE 8 Composition Mixing rate (wt %) N-Acetylhydroxyproline 20Chondroitin 20 Lactose 20 Calcium lactate 10 Magnesium stearate 25Calcium carbonate  5

EXAMPLE 5

A dog food is prepared according to the composition shown in Table 9.

TABLE 10 Composition Content (g) N-Acetylhydroxyplorine 30 Chondroitin10 Pinedex #3 (Matsutani Chemical Industry) 49 Ferric pyrophosphate(iron source) 0.1 Phoscal EF (Calcium source: Nikko Fine Products) 1.0Vitamin mixture (Merck) 1.0

The above mixture (20 g) is dispersed in 180 ml of water to prepare adrink.

EXAMPLE 7

A soft drink (10 bottles) is prepared from the ingredients shown inTable 11 below.

TABLE 11 Composition Content (g) N-Acetylhydroxyproline 30 Chondroitin10 Vitamin C 1 Vitamin B₁ 0.005 Vitamin B₂ 0.01 Vitamin B₆ 0.025 Liquidsugar 150 Citric acid 3 Flavor 1

Water is added to make the volume of 1000 ml.

EXAMPLE 8

A tea drink (1000 ml) is prepared by extracting the ingredients shown inTable 12 below with 1000 ml of water.

TABLE 9 Mixing rate Composition (wt %) N-Acetylhydroxyproline 0.5Glucosamine 0.5 Meat meal 35.0 Corn starch 40.0 Chicken extract 5.0Yeast extract 5.0 Vegetable oil and fat 5.0 Calcium lactate 1.0 Sodiumchloride 1.0 Sodium hydrogenphosphate 0.5 Magnesium carbonate 0.5Ferrous sulfate 0.1 Vitamin B₁ 0.0005 Vitamin B₂ 0.0005 Vitamin E 0.001Niacin 0.005 Vitamin A 2000 IU Vitamin D  150 IU Moisture 6.3

EXAMPLE 6

A drink is prepared from the ingredients shown in Table 10 below.

TABLE 12 Composition Content (g) N-Acetylhydroxyproline 30 Chondroitin10 Tea leaves 15

EXAMPLE 9

Cookies (30 pieces) are prepared from the ingredients shown in Table 13below according to a conventional method.

TABLE 13 Composition Content (g) N-Acetylhydroxyproline 10 Glucosamine10 Chondroitin 10 Soft flour 100 Starch 74 Water 14 Baking powder 2teaspoonfuls Salt ½ teaspoonful Egg one Butter 80 Milk 2 tablespoonfulsHoney small amount

EXAMPLE 10

A loaf of bread (4 pounds) is prepared from the ingredients shown inTable 14 below according to a conventional method.

TABLE 14 Composition Content (g) N-Acetylhydroxyproline 15 Glucosamine15 Strong flour 1000  Sugar 50 Salt 20 Skim milk 20 Shortening 60 Yeast(fresh) 30 Yeast food  1 Water 650 

EXAMPLE 11

Chewing gum (30 pieces) is prepared from the ingredients shown in Table15 below according to a conventional method.

TABLE 15 Composition Content (g) N-Acetylhydroxyproline  1 Glucosamine 1 Gum base 25 Sugar 63 Starch syrup 10 Flavor  1

EXAMPLE 12

Candies (20 pieces) are prepared from the ingredients shown in Table 16below according to a conventional method.

TABLE 16 Composition Content (g) N-Acetylhydroxyproline 1 Glucosamine 1Sugar 80 Starch syrup 20 Flavor 0.1

EXAMPLE 13

Marmalade is prepared from the ingredients shown in Table 17 belowaccording to a conventional method.

TABLE 17 Composition Content (g) N-Acetylhydroxyproline  5 Chondroitin 5 Summer orange peel 500 Sugar 200 Summer orange juice squeezed fromone orange

EXAMPLE 14

Hard capsules (360 mg/capsule) are prepared from the ingredients shownin Table 18 below according to the following method.

TABLE 18 Composition N-Acetylhydroxyproline 250 Glucosamine 250 Lactose 60 Corn starch  30 Hydroxypropyl cellulose  20

N-Acetylhydroxyproline (250 mg) and 250 mg of glucosamine are mixed with60 mg of lactose and 30 mg of corn starch, to which an aqueous solutionof 20 mg of hydroxypropyl cellulose is added. The mixture is kneaded andthen granulated according to a conventional method using an extrudinggranulator. The resulting granules are packed in gelatin hard capsules.

EXAMPLE 15

Soft capsules (170 mg/capsule) are prepared from the ingredients shownin Table 19 below according to the following method.

TABLE 19 Composition Content (mg) N-Acetylhydroxyproline  25 Glucosamine 25 Soybean oil 120

N-Acetylhydroxyproline (25 mg) and 25 mg of glucosamine are mixed with120 mg of soybean oil. The mixture is packed in soft capsules accordingto a conventional method using a rotary dies automatic molding machine.

EXAMPLE 16

Tablets of 8 mm in diameter and 200 mg in weight each are prepared bymixing the ingredients according to the composition shown in Table 20below and tableting the resulting mixture using a tableting machine(Hata Seisakusho, HT-AP15SS-U).

TABLE 20 Mixing rate Composition (wt %) N-Acetylhydroxyproline 20Lactose 40 Calcium lactate 10 Magnesium stearate 25 Calcium carbonate 5

On the other hand, tablets of 8 mm in diameter and 200 mg in weight eachare prepared by mixing the ingredients according to the compositionshown in Table 21 below and tableting the resulting mixture using atableting machine (Hata Seisakusho, HT-AP15SS-U).

TABLE 21 Mixing rate Composition (wt %) Glucosamine 20 Lactose 40Calcium lactate 10 Magnesium stearate 25 Calcium carbonate 5

Furthermore, tablets of 8 mm in diameter and 200 mg in weight each areprepared by mixing the ingredients according to the composition shown inTable 22 below and tableting the resulting mixture using a tabletingmachine (Hata Seisakusho, HT-AP15SS-U).

TABLE 22 Mixing rate Composition (wt %) Chondroitin 20 Lactose 40Calcium lactate 10 Magnesium stearate 25 Calcium carbonate 5

These tablets are packed in separate plastic containers together withsilica gel, respectively and tightly sealed. These plastic containersare packed in the same paper box.

INDUSTRIAL APPLICABILITY

According to the present invention, it is possible to provide apharmaceutical, a food and drink, a food additive, an animal feed and afeed additive that have an effect on the prevention or the treatment ofarthritis, and a method for preventing or treating arthritis in humansor non-human animals using them.

1. A pharmaceutical composition which comprises an N-acylatedhydroxyproline derivative or a salt thereof, an amino sugar or a saltthereof, and a glycosaminoglycan or a salt thereof.
 2. Thepharmaceutical composition according to claim 1, wherein saidhydroxyproline is selected from the group consisting ofcis-4-hydroxy-L-proline, cis-4-hydroxy-D-proline,cis-3-hydroxy-L-proline, cis-3-hydroxy-D-proline,trans-4-hydroxy-L-proline, trans-4-hydroxy-D-proline,trans-3-hydroxy-L-proline and trans-3-hydroxy-D-proline.
 3. Thepharmaceutical composition according to claim 1 or 2, wherein the acylmoiety of said N-acylated hydroxyproline derivative is an acyl grouphaving 2–23 carbon atoms.
 4. The pharmaceutical composition according toclaim 3, wherein said N-acylated hydroxyproline derivative isN-acetylhydroxyproline.
 5. The pharmaceutical composition according toclaim 4, wherein said amino sugar is glucosamine or a salt thereof. 6.The pharmaceutical composition according to claim 5, comprising saidglycosaminoglycan or salt thereof, wherein said glycosaminoglycan ischondroitin sulfate or a salt thereof.
 7. A method for treatingarthritis, comprising administering the pharmaceutical compositionaccording to claim 6 to a patient in need thereof.
 8. The methodaccording to claim 7, wherein said arthritis is rheumatoid arthritis. 9.A food, drink or an animal feed which comprises an N-acylatedhydroxyproline derivative or a salt thereof, an amino sugar or a saltthereof, and a glycosaminoglycan or a salt thereof.
 10. The food, drinkor the animal feed according to claim 9, wherein said hydroxyproline isselected from the group consisting of cis-4-hydroxy-L-proline,cis-4-hydroxy-D-proline, cis-3-hydroxy-L-proline,cis-3-hydroxy-D-proline, trans-4-hydroxy-L-proline,trans-4-hydroxy-D-proline, trans-3-hydroxy-L-proline andtrans-3-hydroxy-D-proline.
 11. The food, drink or the animal feedaccording to claim 9 or 10, wherein the acyl moiety of said N-acylatedhydroxyproline derivative is an acyl group having 2–23 carbon atoms. 12.The food, drink or the animal feed according to claim 11, wherein saidN-acylated hydroxyproline derivative is N-acetylhydroxyproline.
 13. Thefood, drink or the animal feed according to claim 12, wherein said aminosugar is glucosamine or a salt thereof.
 14. The food, drink or theanimal feed according to claim 13, wherein said glycosaminoglycan ischondroitin sulfate or a salt thereof.
 15. A method for treatingarthritis in mammals, which comprises administering a compositioncomprising (I) and (II): (I) an-N-acylated hydroxyproline derivative ora salt thereof, (II) at least one of (i) an amino sugar or a saltthereof, and (ii) a glycosaminoglycan or a salt thereof to a patient inneed thereof.
 16. The method according to claim 15 wherein the patientis a human.
 17. The method according to claim 15 or 16 wherein saidcomposition is administered orally.
 18. The method according to claim 15or 16, wherein said composition comprises both (i) said amino sugar orsalt thereof and (ii) said glycosaminoglycan or salt thereof.
 19. Themethod according to claim 17, wherein said composition comprises both(i) said amino sugar or salt thereof and (ii) said glycosaminoglycan orsalt thereof.